Phospholipase B (PLB) enzymes comprise what is perhaps the most poorly understood class of phospholipases in terms of biological functions. Although several PLB isozymes have been identified in genetically tractable yeasts, their physiological functions are largely unknown. We have elucidated a role for a newly identified member of the fungal PLB family, Plbl, as an essential mediator of osmotic stress response in the fission yeast, Schizosaccharomyces pombe, plb 1delta mutants exhibit mitotic and cytokinesis defects under conditions of osmotic stress that bear resemblance to defects observed in proteasome-defective mutants of S. pombe. They are also able to mate on rich media, a phenotype characteristic of mutants defective in function for the cAMP pathway. These phenotypes are distinct from those caused by loss of function of previously characterized stress response pathways in S. pombe, suggesting that Plb1 regulates a novel pathway. Consistent with the phenotypes caused by loss of Plb1 function, a screen for multi-copy suppressors of the osmosensitive growth defect of plb1 mutant cells resulted in the isolation of genes encoding activators of adenylate cyclase, a 26S proteasome subunit (pra7), and several genes encoding novel proteins, several of which are conserved in mammals. We have determined that the cAMP pathway, like PIb1, is essential for survival of S. pombe cells in hyperosmotic media and that loss of function of the cAMP pathway is rescued by several multi-copy suppressors of plb1delta, including pra7. Our findings lead us to hypothesize that PIb1 functions, in part, to regulate cell cycle progression under conditions of osmotic stress through a novel cAMP and 26S proteasomemediated pathway. We will use the power of yeast genetics, in combination with molecular and biochemical approaches, to further characterize the Plb1-dependent stress response pathway(s) in S. pombe. Our Specific Aims are to: (1) define the enzymatic activities and subcellular localization of Plb1 and determine whether they are altered in response to osmotic stress, (2) establish functional relationships between Plb1 and previously characterized osmotic stress response pathways in fission yeast, (3) investigate roles for the cAMP pathway and 26S proteasome in mediating Plb1-dependent stress response functions, and (4) determine whether other multicopy suppressors of plb1? encode components of the PIb1-dependent stress response pathway. We anticipate that our proposed studies will provide insights into novel mechanisms of stress-induced signal transduction and cell cycle regulation that are likely to be conserved in higher organisms. The proposed project is relevant to several human diseases, including cardiovascular, inflammatory, neurodesenerative, and neoplastic diseases.